We use a variety of experimental systems to address fundamental questions relating to chromatin structure, transcriptional regulation of genetic activity, and differentiation and development. Several studies have been carried out using a method which allows isolation of unique yeast genes, as chromatin, in the expressed and repressed states. We have found that the chromatin structure of the HSP26 gene is essentially identical in the genomic, single copy, and the plasmid, amplified, forms. Surprisingly, the structure is unaffected by heat shock induction of transcription. This suggested that the trans-acting regulatory factors involved in heat shock gene expression are constitutively associated with the gene; minichromosome isolation and examination of proteins present support this hypothesis. Other studies of yeast plasmid chromatin involve the 5S rRNA gene and genes regulated by the MATa locus. We have studied the role of chromatin structure in transcription using the 5S rRNA gene of Xenopus. The gene, or variants, was associated with histones and transcribed in egg, oocyte, or germinal vesicle extracts. A novel method for evaluating the role of positioned nucleosomes was developed to further define the role of chromatin structure in transcriptional regulation. Our data suggest that nucleosome formation inhibits binding of polymerase and TFIIIA. Elongation of transcription is partially inhibited by the presence of a nucleosome. Differences in nucleosomes may relate to transcription through the histone complex; yeast and chicken histones constrain DNA to very different extents. This is of interest due to the fact that yeast chromatin is about 10 times more actively transcribed than that of most larger eukaryotic cells.